This invention relates to gene therapy using transporter systems for delivering nucleic acid into a cell.
Recombinant retroviral vectors have been used for delivery of genes to cells of living animals. Retroviral vectors permanently integrate the transfered gene into the host chromosal DNA. Studies have demonstrated that retroviral vectors can be transduced into liver cells in vivo by direct injection of the virus into the parenchyma after carbon tetrachloride treatment. Kaleko et al., Human Gene Therapy, Vol. 2, pp. 27-32 (1991). Analysis of transduced liver cells indicated a transduction frequency of about 1 out of 160 liver cells. Id.
Retroviral vectors can be transduced into rat hepatocytes after partial hepatectomy followed by a surgical procedure involving isolation and perfusion of the liver with a viral vector. Poorman et al., Arterioscl. Throm., Vol. 11, p. 1413A (1991); Hobbs et al., Annu. Rev. Genet., Vol. 24, pp. 133-70 (1990). The transduced hepatocytes from the above studies were detectable six months after gene transduction. Id.
Another virus used for gene delivery is adenovirus. It has been developed as a means for gene transfer into epithelial derived tissues. Stratford-Perricaudet et al., Hum. Gene. Ther., Vol. 1, pp. 241-256 (1990); Gilardi et al., FEBS, Vol. 267, pp. 60-62 (1990); Rosenfeld et al., Science, Vol. 252, pp. 431-434 (1991). Vectors have been constructed in which the 35 Kb genome in the E3 and E1 regions have been deleted, such that recombinant gene constructs can be inserted into the adenovirus vector. Id. Since adenovirus has a natural tropism for the lung epithelium it was used for gene transfer into this tissue. Gilardi et al., FEBS, Vol. 267, pp. 60-62 (1990); Rosenfeld et al., Cell, Vol. 68, pp. 143-155 (1992).
Recombinant adenoviral vectors have the advantage over retroviruses of being able to transduce nonpoliferating cells as well as an ability to produce purified high titer virus. Studies using an adenoviral vector to deliver genes to liver demonstrated that mouse hepatocytes can be transduced in vivo with the vector. Stratford-Perricaudet, Hum. Gene Ther., Vol. 1, pp. 241-256 (1990). Use of adenoviral-mediated transfer of orninthine transcarbamylase cDNA allowed the transfer of enzyme activity to the mouse liver. These studies resulted in phenotypic correction of enzyme deficiency. Id. Other studies have demonstrated human .alpha.-1-antitrypsin production from rat liver after transduction with a recombinant adenoviral vector. Jaffe et al., Nature-Genetics, Vol. 1, pp. 372-378 (1992). These studies determined that 1% of hepatocytes were transduced in vivo. Id.
In addition to retroviral-mediated gene delivery, a more recent means for DNA delivery has been receptor-mediated endocytosis. Endocytosis is the process by which eucaryotic cells continually ingest segments of the plasma membrane in the form of small endocytotic vesicles. Alberts et al., Mol. Biol. of Cell, Garland Publishing Co., New York, 1983. Extracellular fluid and everything dissolved in it becomes trapped in the vesicle and is ingested into the cell. Id. This process of bulk fluidphase endocytosis can be visualized and quantified using a tracer such as enzyme peroxidase introduced into the extra-cellular fluid. Id. The rate of constitutive endocytosis varies from cell type to cell type.
Endocytotic vesicles form in a variety of sizes and shapes and are usually enlarged by fusing with each other and/or with other intra-cellular vesicles. Stryer, Bioch., Freeman and Co., New York (1988). In most cells the great majority of endocytotic vesicles ultimately fuse with small vesicles called primary lysosomes to form secondary lysosomes which are specialized sites of intracellular digestion. Id. The lysosomes contain a wide variety of degradative enzymes to digest the macromolecular contents of the vesicles. Silverstein, et al., Ann. Rev. Biochem., Vol. 46, pp. 669-722 (1977); Simioesa, et al., J. Cell Biol., Vol. 64, pp. 586-607 (1975).
Many of the endocytotic vesicles are coated and are formed by invagination of coated regions of the plasma membrane called coated pits. Coated pits and vesicles provide a specialized pathway for taking up specific macromolecules from the extracellular fluid. This process is called receptor-mediated endocytosis. Goldstein et al., Nature, Vol. 279, pp. 679-685 (1979); Pearse, et al., Annu. Rev. Biochem., Vol. 50, pp. 85-101 (1981); Pastan, et al., Annu. Rev. Physiol., Vol. 43, pp. 239-250 (1981). The macromolecules that bind to specific cell surface receptors are internalized via coated pits. Goldstein, supra. Receptor-mediated endocytosis is a selective mechanism enabling cells to ingest large amounts of specific ligands without taking in correspondingly large amounts of extra-cellular fluid. Goldstein, supra.
One such macromolecule is low density lipoprotein ("LDL"). Numerous studies have been performed involving LDL and the receptor-mediated endocytotic pathway. In addition to LDL, many other cell surface receptors have been discovered to be associated with coated pits and receptor-mediated endocytosis. Pastan et al., Annu. Rev. Physiol., Vol. 43, pp. 239-250 (1981). For example, studies have analyzed the hormone insulin binding to cell surface receptors and entering the cell via coated pits. Stryer et al., Biochemistry, Freeman & Co., New York (1988); Alberts et al., Molecular Biology of the Cell, Garland Publishing, New York (1983). In addition, it has been determined that some cell surface receptors associate with coated pits only after ligand binding. Pastan, supra.
Taking advantage of receptor-mediated endocytosis, the asialoglycoprotein receptor has been used in targeting DNA to HepG2 cells in vitro and liver cells in vivo. Wu et al., J. Biol. Chem., Vol. 262, pp. 4429-4432 (1987); Wu and Wu, Bio., Vol. 27, pp. 887-892 (1988); Wu and Wu, J. Biol. Chem., Vol. 263, pp. 14621-14624 (1988); Wu et al., J. Biol. Chem., Vol. 264, pp. 16985-16987 (1989); Wu et al., J. Biol. Chem., Vol. 266, pp. 14338-14342 (1991). These studies used asialoorosomucoid covalently linked to polylysine with water soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide or with 3'(2'pyridyl-dithio)propionic acid n-hydroxysuccinimide ester. Polylysine in the studies above bound DNA through ionic interaction. The DNA was ingested by endocytosis.
Other studies have utilized transferrin and the transferrin receptor for delivery of DNA to cells in vitro. Wagner et al., P.N.A.S., Vol. 87, pp. 3410-3414 (1990). These studies modified transferrin by covalently coupling transferrin to polylysine. Id. The polylysine interacted ionically with DNA. Delivery of DNA occurred to cells through the transferrin receptor. Such analyses were performed in vitro. Id. Cotten et al., P.N.A.S., Vol. 87, pp. 4033-4037 (1990); Zenke et al., P.N.A.S., Vol. 87, pp. 3655-3659 (1990).
In addition to DNA, macromolecules can also be delivered by receptor-ligand systems. Leamon et al., P.N.A.S., Vol. 88, pp. 5572-5576 (1991); Leamon et al., J. Biol. Chem., Vol. 267, pp. 24966-24971 (1992). In particular these studies have involved the folate receptor, an anchored glycosylphosphatidyl protein, which is excluded from coated pits and cycles in and out of the cells by caveolae. Hill and Dessler., Science, Vol. 252, pp. 410-414 (1991). This uptake mechanism has been called potocytosis. Id. Folate conjugated enzymes have been delivered into cells through this receptor system and retained activity for at least 6 hours. Leamon et al., P.N.A.S., Vol. 88, pp. 5572-5576 (1991). Folate receptors have limited tissue distribution and are overexpressed in several malignant cell lines derived from many tissues. Weitman et al., Cancer Res., Vol. 52, pp. 3396-3401 (1992); Weitman et al., Cancer Res., Vol. 52, pp. 6708-6711 (1992); Campbell, Cancer Res., Vol. 51, pp. 5329-5338 (1991); Coney, Cancer Res., Vol. 51, pp. 6125-6132 (1991). Other studies have also used biotin or folate conjugated to proteins by biotinylation for protein delivery to the cell. Low et al., U.S. Pat. No. 5,108,921.
DNA and macromolecule delivery is hindered by lysosomal degradation. Studies have analyzed the endosomal/lysosomal degradation process. It has been determined that organisms which are internalized via receptor-mediated endocytosis or receptor:ligand systems, like viruses and other microorganisms, escape lysosomal degradation in order to function. The entry mechanism of some viruses have been studies extensively. For some viruses outer membrane proteins have been demonstrated to be important for endosomal escape. Marsh et al., Adv. Virus Res., Vol. 36, pp. 107-151 (1989). Other studies have focused on methods to prevent lysosomal degradation. These studies have used substances which pertubate endosomal/lysosomal function. Mellmann et al., Ann. Rev. Biochem., Vol. 55, pp. 663-700 (1986). These substances have only been used in vitro. In addition, studies show that the entire virus-shell is necessary for efficient endosomal lysis. Marsh et al., Adv. Virus Res., Vol. 36, pp. 107-151 (1989). Studies have also demonstrated that adenovirus will enhance transferrin-polylysine mediated gene delivery. Curiel P.N.A.S., Vol. 88, pp. 8850-8854 (1991).
A number of bacteria are also internalized via receptor-mediated endocytosis and are liberated from the endosome by production of toxins. These toxins lyse the endosomal membrane. Moulder, Microbiol. Rev., Vol. 49, pp. 298-337 (1985). Listeria monocytogenes produce a membranolytic toxin called listeriolysin. Cossart et al., Mol. Biol. Med., Vol. 6, pp. 463-474 (1989); Tilney et al., J. Cell Bio., Vol. 109 pp. 1597-1608 (1989). Studies have shown that no other cofactors are needed for endosomal escape of Listeria monocytogenes. Bielecki et al., Nature Vol. 345 pp. 175-176 (1990).
The listeriolysin toxin forms pores in membranes which contain cholesterol. These pores are large enough for macromolecules like immunoglobulins to pass. Ahnert-Hilger et al., Mol. Cell Biol., Vol. 31 pp. 63-90 (1989); Geoffroy et al., J. of Bacteriol., Vol. 172, pp. 7301-7305 (1990).